Biology

n

Endodontic Biology:Towards

Regenerative Endodontics

Ove A. Peters, DMD MS PhDDiplomate, American Board of Endodontics

January 17, 2013Seattle WAWashington State Association of Endodontists

Biology

n

Biology

n How I Got There...

Biology

opeters@pacific.edu

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

n Well We Will See….

Biology

Goals§ To review contemporary endodontics ! -!biologic foundational knowledge! -!clinical applications

§ To give my view of the evidence! -!context, literature and clinical strategies! -!where we may be going

§ To provide a range of information! -!confirmatory elements! -!challenging parts, references

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

n

Engineering Biology

Endodonticsn

Biology

Case: Sinus Tract (MB, 04)

Pre-operativeclinical situation

After 2wks ofCa(OH)2 dressing

Biology

Pre-operativeradiographs

Completedtreatment

Workinglength

Case: Sinus Tract (MB, 04)

Biology

Pre-operativeradiographs

4yrfollow-up

Workinglength

Chronic Perirad. Abscessn

Biology

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

Was Surgery Indicated?§ Diagnosis: ! -! radicular cyst! -!significant morbidity

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

The Endodontic Disease

§ Microbial etiology of periradicular pathosis ! -!germ-free rats do not develop periapical lesions! -! teeth with lesions harbor bacteria in their canals

Kakehashi 1965, Sundqvist 1976

§ Therapeutic concepts require a disease ! -!endodontic therapy adresses prevention or re-! ! solution of periradicular inflammation

Ørstavik 1999, Trope 2003

§ Reduction of bacteria via endodontic therapy! -!chemo-mechanical preparation

Byström 1981

Sjögren 1997

§ Prognosis depends on antimicrobial efficacy! -!positive culture correlates with less healing

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

§ Yes, but...! -!healing depends! ! on many factors! -!defining and ! ! detecting healing! ! is even more! ! complex

Successful Disinfection

Whitworth 2000

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

Immune Response

§ Acquired (specific) immunity! - needs prior contact with antigen to be effective

- once activated, very powerful- relatively young

§ System with multiple interactions! - significantly researched because of implications

- new and powerful tools are available- will be significant in the near future: regeneration

§ Innate (unspecific) immunity! - early and drastic response mounted

- cellular, humoral components- developmentally old

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

n

Biology

n

Review

Interactions between immune system and

mesenchymal stem cells in dental pulp and

periapical tissues

J. G. Leprince1,2,3, B. D. Zeitlin

4, M. Tolar5 & O. A. Peters1

1Department of Endodontics, Arthur A. Dugoni School of Dentistry, University of the Pacific, San Francisco, CA, USA;2School of

Dentistry and Stomatology, Universite catholique de Louvain, Brussels,3CRIBIO (Center for Research and Engineering on

Biomaterials), Brussels, Belgium;4Department of Biomedical Sciences, Arthur A. Dugoni School of Dentistry, University of the

Pacific, San Francisco, CA; and5Department of Orthodontics, Arthur A. Dugoni School of Dentistry, University of the Pacific, San

Francisco, CA, USA

Abstract

Leprince JG, Zeitlin BD, Tolar M, Peters OA. Interactions

between immune system and mesenchymal stem cells in dental

pulp and periapical tissues. International Endodontic Journal.

The recent isolation and characterization of mesenchy-

mal stem cells (MSCs) in dental tissues constitutes a

major step forward in the development of new treat-

ment strategies. MSCs are essential for dental pulp

repair and the success of regenerative endodontic

procedures. It is important to understand that immune

cells and cytokines can affect stem cell function, which

can impact their healing potential. On the other hand,

stem cells are immunoprivileged and have the ability to

modulate immune and inflammatory responses, which

can be utilized to improve treatments outcome. This

review addresses both aspects of this interaction and

suggests that any change on both sides can tip the

balance in favour of either persistence of inflammation

or healing. Finally, the therapeutic relevance of the

interaction between MSCs and immune system relative

to current treatments is discussed, and future research

and treatment perspectives are suggested.

Keywords: cytokines, growth factors, immune sys-

tem, immunosuppression, inflammation, mesenchymal

stem cells, periapical lesion, pulp capping.

Received 16 October 2011; accepted 8 February 2012

Introduction

The recent isolation and characterization of mesen-

chymal stem cells (MSCs) in dental tissues constitutes a

step forward in the development of alternate treatment

strategies. MSCs are cells capable of self-renewal and

differentiation in vitro and in vivo into several tissues of

mesenchymal origin, for example bone, cartilage and

adipose tissue (Deans & Moseley 2000, Dominici et al.

2006, Le Blanc 2006). These possibilities have gener-

ated hope and opened a range of new research and

therapeutic perspectives (Zandstra & Nagy 2001). In

addition to these characteristics, MSCs are also char-

acterized by their adherence to plastic culture surfaces

(Dominici et al. 2006). Typical for MSCs is the expres-

sion of specific surface antigens, for example CD29,

CD73, CD90, CD105, in parallel with the absence of

others, like CD34 or CD45 (Dominici et al. 2006).

Several types of dental MSCs have been described, i.e.

dental pulp stem cells (DPSCs), stem cells from human

exfoliated deciduous teeth (SHEDs), stem cells from the

periodontal ligament (PDLSCs), progenitor cells from

the dental follicle (DFPCs) and stem cells form the

apical papilla (SCAPs) (Huang et al. 2009, Rodriguez-

Lozano et al. 2011). Figure 1 illustrates the character-

ization of cultured SCAPs, based on their surface

markers.

Correspondence: Julian Gregoire Leprince, School of Dental

Medicine and Stomatology, Universite catholique de Louvain,

Avenue Hippocrate, 10/5721, Brussels 1200, Belgium

(e-mail: julian.leprince@uclouvain.be).

doi:10.1111/j.1365-2591.2012.02028.x

ª 2012 International Endodontic Journal

International Endodontic Journal1

§ 2011! -! review! -!MSCs, other stem cells are impacted by inflammation - MSCs, other stem cells are immunomodulatory

Biology

n

Biology

Biology ~ Relevance

§ To treat disease appropriately! - currently, endodontic therapy relates to canal disinfection

- the microorganisms are well understood but not healing

§ Future: regenerative endodontics! - clearly, all involved agree that future therapy concepts

have to involve regeneration as opposed to repair- for that to happen, mechanisms need to be understood

§ Interrelation between oral disease and health! - correlation between oral and cardiovascular health? - mechanistic treatment no longer feasible

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

Implants§ Apical lesions have been described! - typical periimplantitis is marginal but occasionally

lesion around the implant apex appear

§ Can teeth with lesions can affect implants?! - apical lesion growth may encompass implant

- implant may be “infected” Sussman 1993

§ Can implants affect adjacent teeth?! - during insertion, a vital tooth may be damaged

- a root-canal treated tooth may be infected during insertion, not clear that it occurs

Margelos 1995

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

n Apical Lesions & Implants

LC, 08; 1m follow-up, case courtesy A. Nattestad

n

Biology

n Clinical Assessment of Pulpal Pathosis

Cold Testing

Normal pulp,Reversible

pulpitis

Irrev. pulpitis,DD: dentin

hypersensitivity,partial pulp necrosis

Necrosis,mineralization,

internal resorption,trauma,

previously initiated Therapy

Pulp status

Normal ormomentary sensation

exaggerated,longer

sensation

no response

n

Biology

n

Biology

n

§ Material & Methods! -!31 patients participated; 19 had irreversible pulpitis based on clinical tests: lingering and spontaneous pain, no PARL; 12 had no symptoms but needed deep fillings replaced - dentinal fluid was picked up over 2mins with a filter paper - a sensitive fluorescent assay was used to detect MMP-9 content - non-parametric statistics were used

J Endod 2011, 37:1293-1295

Biology

n

J Endod 2011, 37:1293-1295

MMP-9 Content

Biology

Pulp Testing Study

§ Clinical data! - vital pulp therapy is currently not very predictable - may provide an alternative to pulp regeneration

§ Sensitivity / Specificity! - 3 symptomatic pulps were actually necrotic

- better tests are needed to enhance outcomes

n Introduction

n Physiology

n Microbiology

n Regeneration

§ Conclusion! - significant development potential - multivariate methods needed: microarrays, etc.

J Endod 2011, 37:1293-1295

Biology

n

Biology

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

TGFβ

§ Experimental data! - increases expression of genes associated with mineralization, cell differentiation

n Introduction

n Physiology

n Microbiology

n Regeneration

§ Clinical conclusion - TGFβ is a relevant molecule in repair/regeneration - liberation of sequestered TGFβ from dentin is important

§ Localization! - radicular dentin (accessible in RCT)

- coronal dentin (accessible below carious lesion)Smith 1995

Biology

n

Graham et al, Biomaterials 2006, 27:2865-2873

ECM Extracted From Dentin

EDTA

Ca(OH)2

Biology

n

Biology

Mixed Infectionbacteria

fungi

virus

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

Successful Decontaminationn Introduction

n Physiology

n Microbiology

n Regeneration

Biology

How Do Microbes Succeed

§ They can persist in vegetative forms ! -!dormant bacteria remain after systemic or! ! low-dose antibiotics

Molander 2003, Figdor 2003

§ They multiply and have well adapted mutations! -!bacteria can escape ecological pressure through! ! many strategies, one of which is mutation

Dahlén 1992

§ They can co-operate to escape host defence! -!biofilms allow bacteria to protect themselves

Byström et al 1981

Jiang 2003

§ They co-operate to increase virulence! -!LPS and peptidoglycans co-stimulate osteoclasts

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

Current Microbiology

§ Sample acquisition - ideally, 100% yield - clinically difficult

§ Using molecular methods - viability of the detected species not clear - finer methods will detect more species

n Introduction

n Physiology

n Microbiology

n Regeneration

§ Conclusion - bacterial composition perhaps similar comparing primary endodontics to retreatment flora!

Hong et al 2013

Biology

E. faecalis?

S. Erlandsen, U Minn

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

Bacteria in Dentinal Tubulesn Introduction

n Physiology

n Microbiology

n Regeneration

Biology

Extraradicular Infection

§ Upside: treatment rationale! -! intraradicular infection is much more accessible! -!non-surgical retreatment has high success

§ Consensus: infection is primarily inside canal! -!good evidence from SEM and TEM studies! -!culturing is prone to false positives

§ Anatomy: where does the canal end?! -!bacteria may lodge in apical canal parts, close to! ! the “end“ of the root canal

§ When is extraradicular infection present ! -!acute abscess, chronic abscess! -!special bacteria: Actinomyces, Propionibacterium

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

n

Sundqvist und klinisches Bild mit “guter” WF

Nair 2004

n

Biology

How Do Bacteria Attack?§ With virulence!! -!They come in VERY big numbers! -!They have several unpleasant abilities

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

How Do Bacteria Attack?§ With strategy!! -!They arrange in biofilms

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

How Do Bacteria Attack?§ With strategy!! -!They arrange in biofilms (movie from Singh et al, Nature 417, 2002)

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

n Introduction

n Physiology

n Microbiology

n Regeneration§ What is it?! - return of a vital response after nonvital response

- in this context: reconstitution of functional pulp

§ What are the goals and benefits! - deposition of hard tissue for immature teeth

- immunologically competent functional tissue

§ What are the indications! - currently: teeth with large apical foramina

- research is underway to extend the spectrum

Regenerative Endodontics

Biology

§ Stimulation vs. delivery! - removal of cells and tissues, recruitment of un-

differentiated cell populations

Metzger 2007

n Introduction

n Physiology

n Microbiology

n Regeneration

Regenerative Endodontics

§ Stimulation vs. delivery! - autologous cells, apical papilla cells

- stem cells from other sources

Biology

§ Crucial: Blood supply! - VEGF and other key factors

- transportation of bacteria/debris into periapical area

§ Scaffold! - collagen, gel etc.

- fibrin matrix

n Introduction

n Physiology

n Microbiology

n Regeneration

Regenerative Endodontics

Biology

n Introduction

n Physiology

n Microbiology

n Regeneration

§ PreopCase by Dr. Patrick King

§ 9m recall

Regenerative Endodontics

Considerations f

or Regeneration

Procedures

Alan S. Law, DDS,

PhD

Abstract

When pulp tissue becomes

necroticin immatur

e teeth,

the prognosis of

the teeth is com

promised. Disinf

ection

of the root(s) pre

sents several ch

allengesincludin

g diffi-

culties in cleaning

and shapinglarge canals w

ith open

apices,obturati

on of canals with open apices,and

potential root fra

ctures caused by

thin and/or weak

ened

root walls. Regenera

tive endodontic procedu

res may

increasethe prognos

is of the compromised immatur

e

tooth by re-establishment

of a functional pulp

tissue

that fosters continue

d root developmen

t and immune

competency. Thi

s article reviewsthe literatur

e related

to anddiscuss co

nsiderations for r

egenerative endo

dontic

procedures and

how these procedure

s may increase t

he

prognosis for im

matureteeth with necrotic

pulp tissue.

(J Endod2013;39

:S44–S56)

Key Words

Chlorhexidine, c

onsiderations, di

scoloration, imm

ature,

mineraltrioxide

aggregate, necr

otic, outcomes,

radio-

graph, regenerati

ve endodontics,

revascularization

, revi-

talization, sodiu

m hypochlorite, to

oth, triple antibiot

ic

paste

One of thegoals of

endodontics is ke

eping the dentiti

on in a physiolog

ically func-

tional state for the

maintenance of o

ral and systemic h

ealth. Trauma to

the devel-

oping dentition, w

hen it results in p

ulp necrosis and

incomplete root

formation, can

lead to aprematu

re loss of perman

ent teeth, which

may compromise

the dentition.

Teeth with imma

ture apices prese

nt challenges in

cleaningand shap

ing largecanals

with blunderbuss

apices, obturation

of canalswith ope

n apices, and pot

ential root frac-

tures caused by th

in and/or weaken

ed root walls (1–3

). By regeneratin

g tissuein the

canal space of imma

ture teeth, regenerativ

e techniques can help to restore

the

physiologically fu

nctionaldentition

.

The concept of r

evitalizing tissue

in the canal spac

e has been aroun

d for several

decades.A case se

ries by Nygaard-O

stby andHjortdal

in 1971(4) show

ed ingrowth of

fibrous connectiv

e tissue and ceme

ntum in mature teeth w

ith previously ne

crotic pulp

tissue. In1974, M

yers andFountain

(5) reported an in

creasedroot leng

th and calcified

materialin necro

tic canals of mon

key canines with

immature apices

after disinfection

with NaOCl and fil

ling the canals wit

h citratedwhole bl

ood or gel foam.

In 1976,Nevins

et al (6)showed

that the disinfectio

n of necrotic cana

l space inimmatur

e monkey inci-

sors followed by t

he placement of a

gel (containing c

ollagen,calcium

chloride, and di-

potassium hydroge

n phosphate) and

short obturation

with gutta-percha

could result in

‘‘variousforms of

hard andsoft con

nective tissue’’ in

cluding‘‘cement

um, bone, and

reparative dentin

’’ liningthe walls

of the root canal

. Although the findi

ngs of the 3

reportsshowed

the possibility of

creatingnew tissue in

necroticcanal sp

ace, it was

not untilthe past

decade that there

has beenan incre

ased interest in a

pplyingthese

conceptsin clinic

al endodontics.

Two case report

s can becredited

with sparking re

cent interest in r

egenerative

endodontics. The

first, which was b

y Iwaya et al (7), s

howed that NaOCl

and H2O2disin-

fection of necroti

c tissuein an im

mature premolar

followedby canal

medication with

metronidazole an

d ciprofloxacin resulted

in continued root for

mation and clinical

evidenceof rener

vation. The secon

d, by Banchs an

d Trope (8), show

ed a similar

outcomeof contin

ued rootdevelopm

ent andrenervat

ion aftercanal dis

infectionwith

NaOCl and chlorh

exidine and the p

lacement of tripl

e antibiotic paste

(TAP; ie, cipro-

floxacin,metronid

azole, and minoc

ycline). Both of th

ese casereports s

howed 3impor-

tant principles of

regenerative endo

dontic procedur

es:

1. Elimination o

f bacteria from the cana

l system

2. Creation of a

scaffoldfor the in

growth of new tissue

3. Prevention of

reinfection by cr

eating abacteria-

tight seal

Althoughthese 2 c

ase reports show

ed clinical eviden

ce of continued ro

ot growth,

they didnot show

histologyof the ca

nal contents. As a

result, itis not cle

ar what type of

tissue was presen

t. Therehave bee

n several terms u

sed to describe th

e introduction of

new living tissue i

nto the canal spac

e. Theseinclude r

egeneration, reva

scularization, and

revitalization. The

re has been debat

e as to which of th

ese 3 terms (ie, r

evascularization,

revitalization, or

regeneration) is m

ost appropriate t

o describe the ou

tcome ofproce-

dures used to reg

enerate pulp tissu

e (3, 9, 10). The t

erm revascularization

describes the

re-establishment

of the vascular su

pply to existing p

ulp in immature p

ermanent teeth

(11). Revitalizatio

n describes the in

growth of tissue t

hat maynot resem

ble the original

lost tissue (12). E

ndodontic regen

eration is the rep

lacement of ‘‘dam

aged structures,

including dentin

and root structures, a

s well as cells o

f the pulp-dentin

complex’’

(13). More rece

ntly, 2 case reports (14

, 15) have inclu

ded histologic section

s of

teeth that were e

xtractedafter reg

enerativeendodon

tic treatment. The

findingsfrom

these reportsmay give a glimpse

of what may be occurring in other teeth.

Torabinejad and

Faras (14) prese

nted clinical, rad

iographic, and h

istologicfindings

From The Dental Speci

alists, Lake Elmo

, Minnesota; and the

Divisionof Endo

donticsand Graduat

e Endodontics, U

niversity

of Minnesota, School

of Dentistry, Minneapo

lis, Minnesota.

This article is being

published concurre

ntly in Pediatric

Dentistry 2013

;35(2). The articl

es are identical.

Either citation

can be used whe

n citingthis artic

le.

Addressrequests

for reprints to D

r Alan S. Law, T

he Dental

Specialists, 8650

HudsonBouleva

rd, LakeElmo, M

N 55042.

E-mail address:

endolaw@comcast

.net

0099-2399/$ - s

ee frontmatter

Copyright ª 2013 Am

erican Academy

of Pediatric Dent

istry

and American A

ssociation of End

odontists.

http://dx.doi.org/

10.1016/j.joen.2

012.11.019

Injuriesto Permanent De

ntition Symposium

S44 Law

JOE —Volume

39, Number 3S, M

arch 2013

Biology

n

Law 2013

§ Clinical steps! -! indication, prognosis! -!anesthesia - 1st appointment • irrigation • disinfectant paste • scaffold - 2nd appointment • pulp space barrier • definitive filling

Colleagues for

Excellence

Spring 2013

Regenerative Endodontics

Cover artwork: Rusty Jones, MediVisuals, Inc.

Published for the Dental Professional Community by the

American Association of Endodontists.

This issue sponsored by a grant from the AAE Foundation.

www.aae.org/colleagues

Biology

n§ Clinical steps! -! indication, prognosis! -!anesthesia - 1st appointment • irrigation • disinfectant paste • scaffold - 2nd appointment • pulp space barrier • definitive filling

Biology

n

Law 2013

Biology

Considerations§Indication - large apical diameter, young patients

§Diagnosis - pulp necrosis, sinus tracts

§Expected outcomes - favorable / unfavorable

§Clinical steps - disinfection, scaffold, definitive closure

n Introduction

n Physiology

n Microbiology

n Regeneration

Root Length Wall Thickness

Biology

Expected Outcomes§ Apical barrier - radiographically visible, apical stop for fill

Bose 2009

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

Regenerative Endodontics

§ Predictability - treatment outcome may depend strongly on conditions - no clear clinical recommendation for procedure

§ Nature of the deposited hard tissue - strengthening effect not clear - hard and soft tissues are important

Paqué, 2011

§ Conclusion - vital pulp therapy may become attractive? - significant development potential

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

n

J Endod 2011, 37:1499-1503

Histologic Chara

cterization of Engin

eered Tissues

in the Canal Spa

ce of Closed-ap

ex Teethwith Ap

ical

Periodontitis

Jo~ao Eduardo Gomes-Filh

o, PhD,* Paulo

Carvalho Tobias

Duarte, PhD,

* Edilson Ervolin

o, PhD,†

Suely ReginaMogami Bom

fim, PhD,‡ Caio Jos!e Xa

vier Abimussi, M

Sc,‡

Ludmilla Mota da Silva Santos, MSc,* Caroli

na SimonettiLodi, P

hD,*

SandraHelena

PenhaDe Oli

veira,PhD,

† El!oi Dezan, Jr

, PhD,*

and Luciano Tavare

s Angelo Cintra

, PhD*

Abstract

Introduction: T

he aim of this study wa

s to investigate

the capacityof endodon

tic regenerative procedu

res

combining an induced

blood clot, platelet-rich

plasma

(PRP), and bone ma

rrow aspirate(BMA) to regenera

te

dentalpulp in canine

closed-apex necrotic

teeth.

Methods:Apical p

eriodontitis was

inducedin 20 up

per

and lower premolars

of 2 dogs. After bio

mechanical

preparation, enla

rgementto a #60

file, anddisinfect

ion

with a triantibiotic paste for 28 days, th

e roots were

randomly assigned

to 4 treatment grou

ps: blood clot

(BC), BC+ PRP gel, BC

+ BMA gel, andBC + BMA/

PRP gel. Negative controls

were also included. After

a 3-monthfollow-u

p period,the animals

were killed.

Results: Histolo

gic analysisshowed

the presenceof

newly formedvital tis

sues (connective, cement-

like,

and bone-like tissue)

in 23 of the 32 treatedroots

(71.87%). There

was nostatistica

lly significant differ

-

ence betweenthe treatme

nt groups.Conclus

ions:

New vital tissues were formed

and characterized as

connective, cem

entum-like, or

bone-like, but

not as

pulp-liketissue; P

RP and/or BMA did not imp

rove the

tissue ingrowth.

(J Endod2013;-

:1–8)

Key Words

Bone marrowaspirate

, necroticteeth, platelet-

rich

plasma,regenera

tive endodontics, tiss

ue engineering,

vital tissue grow

th

Completerestorati

on of a functional dentin

-pulp complexcannot b

e achieved

by conventional en

dodontictreatmen

t (1, 2). Howev

er, withthe advent o

f

regenerative medicine

, it became possible

to regenerate pulp through

alternative

endodontic regen

erative procedur

es (ERPs) (3). Th

ese procedures a

re basedon the

principles of tissu

e engineering tha

t aim to develop new tissues to

replace lost or m

al-

functioning orga

ns by using a sou

rce of stem cells, a th

ree-dimensional s

caffold for the

growth of these c

ells, andsignaling

molecules (1, 3)

. The most com

mon ERP uses

blood clots and

cell transplantati

on. In anumber

of studies, the in

ductionof a clot

alone did not promote pulp regenera

tion in immature necrotic

dog teeth (4–6),

prompting the in

vestigation of new

ERPs that modify

the clotor do no

t use it at all.

Platelet-rich plasm

a (PRP)has been

investigated in th

e context of hum

an ERPs

(3). A patient treated with PRP showed

the presence of

pulp tissue and a lack of

bone tissue (7). H

owever,no pulp

regeneration was

achievedby inject

ing PRP,dental

pulp stemcells (DP

SCs), ora mixtur

e of bothinto the r

oots of mature do

g teeth without

inducinga clot (8

). Moreover, in a

human subject, i

njectionof PRP a

nd bloodclot

caused an increasein root len

gth and thickness compa

red with that observed with

the induction of

a bloodclot alon

e (9).

Dog pulp regeneration has bee

n achievedby using

stem cell transplantati

on

(10–12). Howev

er, cultivation an

d expansion hav

e been linked to

reducedviability,

selection, and unwante

d reprogramming a

nd/or cellular de

differentiation (13). In

addition, it is ex

pensive,time-con

suming,and associate

d with an increased risk of

infection(14, 15)

. Moreover, as t

he pulpdiminish

es withage, alte

rnative sources

need tobe evalu

ated (12). Bone

marrowmesench

ymal stem cells (M

SCs) have been

used as an alterna

tive source for do

g pulp regeneratio

n (12). The use o

f bone marrow

aspirate(BMA) c

an be a straightfo

rward, low-cost, a

nd fast method wi

th a lowrisk of

contamination (1

6, 17).

An ERP for matur

e necrotic perma

nent teeth has be

en clinically inves

tigated, and

the resolution of

apical radiolucen

cy and regressio

n of clinical sign

s and symptoms

were observed a

t recall appointm

ents (18). Moreo

ver, histologic eva

luation of trans-

plantation of DPS

Cs and/or PRP in

to root canals sh

owed noevidence

of pulptissue

or evenimprove

d tissueingrowth

in mature vital do

g teeth (8). How

ever, noreported

study has aimed

to histologically in

vestigatepulp reg

eneration of clos

ed-apexnecrotic

teeth byusing alt

ernativeERPs. Th

e aim of this study was

to investigate the

synergistic

From the *Departmen

ts of Endodontic

s and†Basic Sc

iences, Aracatuba

School of Dentis

try, Universidade

Estadual Paulist

a, Aracatuba, S

~ao Paulo; and

‡Clinical,

Surgeryand Ani

mal Reproductio

n, Aracatuba Sch

ool of Veterinar

y, Universidade

Estadual Paulist

a, Aracatuba, S

~ao Paulo, Brazil.

Supported by CN

Pq (National Res

earch Council).

Addressrequests

for reprints to D

r Jo~ao Eduardo G

omes-Filho, End

odontics, Aracat

uba School of D

entistry,Universi

dade Estadual P

aulista,R. Jos!e B

onif!acio, 1193

Aracatuba, S~ao

Paulo, Brazil. E-m

ail address: jegom

esfilho@yahoo.c

om.br

0099-2399/$ - s

ee frontmatter

Copyright ª 2013 Am

erican Associatio

n of Endodontist

s.

http://dx.doi.org/

10.1016/j.joen.2

013.08.023

Basic Research—Technolo

gy

JOE — Volume-, Numbe

r -, - 2013

Engineered Tissu

es in Teeth with A

pical Periodontiti

s 1

Biology

n§ Human histology:“The tissues formed in the canal of revitalized human tooth are similar to cementum- or bone-like tissue and fibrous connective tissue.”

Shimizu 2013

Biology

§ Disinfection - no instrumentation - irrigation with 1.5% NaOCl - place Ca(OH)2 or TAP/DAP

First Appointment§ Case selection - post space needed, allergies, compliance?

§ Access

§ Consent - mostly minors - discoloration of the crown possible: TAP, MTA

§ Temporary filling

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

§ Ca(OH)2

- recent data shows less cytotoxic compared to TAP - readily available, place with lentulo spiral

§ Temporary filling - should avoid recontamination - e.g., 4mm layer Cavit plus IRM

Issues§ TAP/DAP - 1:1:1 minocyline, ciprofloxacin, metronidazole - staining due to minocycline, remove or seal dentin

n Introduction

n Physiology

n Microbiology

n Regeneration

Direct Effect of

Intracanal Medicame

nts on Survival o

f Stem

Cells ofthe Apic

al Papilla

NikitaB. Rup

arel, DDS, MS, PhD

,* Fabricio B. Teix

eira, DDS, MS, PhD

,*

Caio C.R. Ferraz, D

DS, MS, PhD,† and

Anibal Dioge

nes, DDS, MS, PhD

*

Abstract

Introduction: R

egenerative end

odonticprocedu

res are

an alternative treatme

nt for immature teeth with

necroticpulps. T

ypically,intracan

al medicaments

such

as tripleantibiot

ic paste(TAP) or

double antibiotic

paste

(DAP) and calcium

hydroxide (Ca[OH]2

) are used for

disinfection. How

ever, their effect

on human stem cells

of the apical pa

pilla (SCAPs) is u

nknown. We hypoth

e-

sized that intracanal m

edicaments at high

concentra-

tions aretoxic to

SCAPs.To test this

hypothesis, a ce

ll

cultureassay was used. Methods:

Briefly,SCAPs

were cultured an

d subjected to ei

ther no drug trea

tment

or various concen

trationsincludin

g TAP, DAP, mod

ified

TAP (ciprofloxacin, m

etronidazole and cefaclor

), Aug-

mentin(Champ

s Pharmacy, San Antonio

, TX), or

Ca(OH)2. Viable

stem cells counts were

obtainedusing

an automated method

of detecting trypan blue dye at

3 days after treatment. Results

: All 4 antibiotics

significantly red

uced SCAP survivalin a concent

ration-

dependent fashion.

Interestingly,

Ca(OH)2was

conducive with SCAP survival

at all concentratio

ns.

Conclusions: Collectiv

ely, ourdata show that high

concentrations

of antibiotics ha

ve a detrimental effec

t

on SCAP survival,whereas

lower concentrations

as

well asCa(OH)2

at all tested con

centrations are c

ondu-

cive with SCAP

survivaland prol

iferation. These

studies

highlight the clinically

important point

that intracanal

medicaments mu

st be used at concentration

s that are

bactericidal whi

le having minimaleffects o

n stem cell

viability. (J Endo

d 2012;38:1372–

1375)

Key Words

Apical papilla, b

iocompatible, ca

lcium hydroxide, denta

l

stem cells, double antibiot

ic, human stem cells of

the

apical papilla, re

generative, triple

antibiotic

Regenerative endo

dontic therapy ha

s become a viabl

e alternative in th

e treatment of

immature teeth w

ith pulpal necros

is. Sinceits incep

tion, several suc

cessful case

reportshave been publishe

d (1–9)with substant

ial advancements in the

understanding of

the biological pri

nciples involved

in the regeneration

of the pulp-

dentin complex,

including the eva

luation of stem cells, gro

wth factors, and s

ignaling

molecules involve

d in thedifferent

iation ofstem cells into

various cell types

(10–12).

Moreover, it has b

ecome evident fr

om preclinical studie

s and clinical cas

e reportsthat

a successful rege

nerativeprocedu

re of a tooth wit

h necrotic pulp r

equiresadequate

disinfection of th

e root canal syste

m.

Clinicalregenera

tive endodontic procedu

res dependprimaril

y on chemical

debridement of the

canal space with

minimalto no me

chanicalinstrume

ntation (1–8).

The most widely used intracanal medicam

ent used in regenerativeendo

dontic/

revascularization

procedures is the mixture

of ciprofloxacin, metronid

azole,

and minocycline, wh

ich is also called triple antibiotic paste (1, 13,

14). This

combination has bee

n shown to be highly efficaciou

s against bacter

ia commonly

found ininfected

root canal system

s in bothin vitro (1

3, 14) and in vivo stu

dies

(1, 3, 5, 9, 15)

. However, other

intracanal medic

aments have been

used including

Ca(OH)2(4); form

ocresol(7); the

combination of m

etronidazole and

ciprofloxacin

(doubleantibioti

c paste)(2); and

the combination

of metronidazole

, ciprofloxacin,

and cefaclor (mo

dified triple antib

iotic paste) (8).

Althoughthe comb

ination of amox-

icillin and clavulo

nic acid(Augmen

tin; Champs Pharm

acy, SanAntonio,

TX) hasnot yet

been used in a pu

blished regenerat

ive endodontic ca

se, it hasbeen fou

nd to beeffective

against 100% of endod

ontic bacteria (16

), and itmay repr

esent animportan

t medica-

ment alternative

for patients witho

ut a history of a p

enicillin-like drug

allergy. Collec-

tively, the antibac

terial effects of th

ese drugs are we

ll known; howeve

r, there is a large

gap in knowledg

e regarding the p

otentialtoxic eff

ect on human mesench

ymal stem

cells. Thus, the a

im of this study was

to evaluate the eff

ect of commonly

used intracanal

medicaments on t

he survival of hum

an stemcells of th

e apicalpapilla (

SCAPs) in vitro.

Materialsand Methods

Patient Recruitment

This study was ap

proved by the Ins

titutional Review

Board of the University

of

Texas Health Scie

nce Center at San

Antonio.After ver

bal andwritten c

ommunication

for informed con

sent, stemcells of t

he apical papilla

were harvested fr

om 2 extracted

immature mandi

bular third molar

s (#17 and #32)

from a 17-year-old fem

ale patient

recruitedfrom the clinic

of the University o

f Texas Health Sci

ence Center at San

Antonio

School of Dentist

ry. The extracted

teeth were imme

diately rinsed in s

terile phosphate-

bufferedsaline (P

BS) andstored in

a container with s

terile PBS until SC

AP harvesting.

Harvesting of SCAP and Cell Cult

ure

SCAPs were obta

ined fromthe harve

sted apical papilla

e by enzymatic dig

estion as

described previo

usly (17). Cells i

n suspension wer

e platedin 10-cm

diameterpoly-D

lysine-treated cult

ure plates (BD Bioscien

ces, Bedford, MA

). The cells form

ed single

coloniesand exhi

bited typical fibro

blast-likemorpho

logy. Cells were a

llowed toexpand

in culture to 70%

to 80% confluency follow

ed by treatment w

ith 0.05%trypsin (

Gibco,

Carlsbad, CA) and

passing the cultur

e to subsequent c

ulture plates. SCA

Ps between the

fifth andeighth p

assageswere use

d in thisstudy.

From the *Departmen

t of Endodontics

, University of T

exas

Health Science C

enter atSan Ant

onio, San Anton

io, Texas; and

†Department of Restorat

ive Dentistry, Pirac

icaba Schoolof

Dentistry, State

University of Ca

mpinas,S~ao Pau

lo, Brazil.

Addressrequests

for reprintsto Dr Anibal

Diogenes,

Department of En

dodontics, Unive

rsity of Texas He

alth Science

Center at San Antonio

, 7703Floyd Curl Dri

ve, SanAntonio

,

TX 78229. E-mail ad

dress: Diogenes

@uthscsa.edu

0099-2399/$ - s

ee frontmatter

Copyright ª 2012 Am

erican Associatio

n of Endodontist

s.

http://dx.doi.org/

10.1016/j.joen.2

012.06.018

Basic Research—Technolo

gy

1372 Ruparelet al.

JOE —Volume

38, Number 10,

October2012

Biology

n§ Toxicity! -!TAP in the currently used way has high concentration - this is toxic to stem cell, impacting proliferation and differentiation - lower concentrations and Ca(OH)2 are preferred

Biology

§ Reaccess and irrigation - EDTA rather than antimicrobials

§ Tissue barrier - collagen app. 3mm below CEJ, MTA fill, alt. GIC

Second Appointment§ Timing - after 3-4 weeks, longer not advisable

§ Anesthesia - no vasoconstrictor

§ Scaffold - overinstrument to create bleeding, alt. collagen plug

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

n

§ Material & Methods! -!SCAP were isolated from third molars and characterized - human root segments were subjected to irrigation with either 17% EDTA; 6% NaOCl/EDTA; EDTA/2%CHX or NaOCl/EDTA/NaOCl/alc/CHX - root segments were filled with SCAP and PRP, cultured for 21 days and then processed for immunohistochemistry - cell viability was determined

J Endod 2011, 37:1109-1115

Biology

n Effect of Irrigation on SCAP

J Endod 2011, 37:1109-1115 A B C D

Biology

§ Radiographic - apical lesion resolves (6-12m) - root wall thickness increase (12-24m) - root lengthening, apical closure (variable)

§ Discoloration - sometimes esthetically compromising

Follow-up§ Clinical - no pain or tissue swelling - between the two primary appointment and at 6m recall

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

§ Research impact - expect to broaden biological knowledge base - will establish molecular methods in the clinic

§ Strategies - vital pulp therapy may become attractive and feasible - significant development potential

What to Expect:§ Public health impact - patient population currently small - extension to mature apices possible?

Verma 2012

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

n§ MTA pulp caps! -!success rate varies from >90% to about 50%

Biology

n

Biology

§ Immature root development - reasonable alternative to RCT - MTA material of choice

§ Strategies - avoiding pulp exposure preferrable - few well-done clinical studies

Vital Pulp Therapy§ Predictable? - for adults depends on pulpal status - tests insufficient: cold, electric

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

Conclusions§ Long-range: two pillars - vital pulp therapy - minimal invasive conventional endodontics

§ Cognitive framework - establish best practices, currently insufficient evidence - socioeconomics and access to care

§ Transition period - gradual R & D for both - special cases: define indications and techniques

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

Conclusions§ Elimination of microbes is key to success ! -!presence of organisms linked to disease symptoms! -!both counts and virulence of colonies are important

§ Efficacy of antimicrobial regime can vary! -!understanding chemical and biological interactions! -!assessing cases and their individual challenges

§ Clinical strategies must vary ! -!wide range of possibilities! -! technical and biological details

n Introduction

n Physiology

n Microbiology

n Regeneration

Biology

A Break?

False Summit, Mt. Tam

n Introduction

n Physiology

n Microbiology

n Regeneration